Broadleaved germinable tree species is referred to a collection of arbor and half-arbor broadleaved tree species capable of regenerating new branches after cutting back, which comprises a variety of the families Salicaceae, Ulmaceae, Leguminosae, Rosaceae, Oleaceae, Betulaceae, Tiliaceae, Labiatae, Simaroubaceae, Aceraceae, Sterculiaceae, Cruciferae, Lauraceae, Plantanaceae, Scrophulariaceae, Theaceae, Meliaceae, Orchidaceae and Fagaceae and the like. Poplar, among the family Salicaceae, is the typical representative of such collection of broadleaved germinable tree species.
Poplar is the most widely-distributed tree species in the world, with the strongest adaptability. It is primarily distributed in the temperate zone and cold temperate zone of 22°-70° north latitude, and in the regions of from low altitude to high altitude of 4800 meters.
The distribution area of poplar natural forest is about more than 20,000,000 hectares (poplar is the dominant species standing in the world natural forest), with about more than 100 species. The countries where poplar is distributed most widely are Russia, China, Canada, USA, Italy and France etc. There is a rich resource of poplars in China with the natural forest accounting for about 3,000,000 hectares and having up to 53 natural species with a wide distribution, which are ranging from Populus suaveolens Fisch and Populus ussuriensis Kom in the most northern regions of Daxing'an and Xiaoxing'an Mountains to Populus yunnanensis Dode in southern regions, and from Populus euphratica Oliv tolerant to drought and salt, to Populus alba Linn, to Populus canescens Smith etc. in areas ranging from east to west in China.
Poplar has two characteristics of early fast-growing and asexual propagation. However, there are two problems in artificial forest of China East and West as follows.
The first problem is that poplar artificial forest of Sanbei shelter forest is 4,000,000 hectares, and ⅖ of fund and labor used in Sanbei shelter forest is for the poplar development annually, which occupies 27% of forest area in the same period. The current serious problem present is: runt trees is more than 1,400,000 hectares; Longicorn beetles and Cossidae cause a great harm, and the counties damaged have been developed from the initial 30 to 240, with about 600,000 hectares damaged. The reasons for this problem are that deterioration of environment, not planting trees in suitable soil; and low level of intensive cultivation.
The second problem is that low productivity of eastern commercial forest, with less than 30% area of the artificial forest being capable of forming real commercial forest. The reasons are single variety, variety ageing, low productivity and low level of intensive cultivation.
Therefore, a method of breeding the plants of a germinable transgenic broadleaved tree species, particularly, those of a transgenic poplar, is needed urgently for the directional improvement of a poplar variety using genetic engineering means and to resolve the ecological problem, such as vegetation restoration etc. and the wood problem.
Plant transgenic technique is mainly by agrobacterium mediated transformation and gene gun mediated transformation. Agrobacterium mediated transformation possess the advantages of easy performance, low cost, high transformation efficiency, good repeatability, being able to introduce a large fragment of DNA, the gene introduced being incorporated with a single copy or low copies, and decreasing the occurrence of gene silence, and it is used most widely and has been used in and extended to dicotyledon economical crops, such as soybean etc. With a deeper comprehension of the mechanism of agrobacterium mediated transformation, the improvement on this transformation method is being made, for example, such as using strains with high invasiveness, super binary vectors, suitable explants, high efficient promoters, suitable co-transformation medium, appropriate selective agent, treatment with higher concentration of phenolic compound, such as Acetosyringone (AS), etc (Kong Y, Zhou G, Wang G, and Wang Y, 2000. Factors affecting the transformation of Agrobacterium tumefaciens and their applications on cereals. Chinese Journal of Applied Ecology, 11(5):791-794). Transgenic plants have been successfully obtained in monocotyledon with important values, such as rice, corn, wheat, barley etc, and specific molecular biological evidences are obtained (Hiei Y, Komari T, Kabo T., Transformation of rice mediated by Agrobacterium tumefaciens., Plant Mol Biol, 1997, 35:205-218; Ishida Y, Saito H, Ohta S. High efficiency transformation of maize (Zea mays L) mediated by Agrobacterium tumefaciens, Nat Biotech, 1996, 14:745-750; Cheng Ming, E. F. Joyce, Pang Shengzhi et al., Genetic transformation of wheat mediated by Agrobacterium tumefaciens, Plant Physiol, 1997, 115:971-980). Now, people devote to widen the scope of monocotyledon used as host of agrobacterium transformation and increase the transformation efficiency, for example, Chai Bao-Feng etc established the technical solution of agrobacterium mediated transformation in monocotyledon Kentucky Bluegrass (CHAI Bao-Feng, LIANG Ai-Hu, WANG Wei, Hu Wei, Agrobacterium-mediated Transformation of Kentucky Bluegrass, Acta Botanica Sinica, 2003, 45(8): 966-973).
But the key for obtaining the plants of a germinable transgenic broadleaved tree species, particularly poplar, is not the gene transformation technique, but is how to regenerate intact plants with high efficiency using the transformed cells. Therefore, the establishment of a high efficiency, practical transformation system is the important bottleneck of obtaining transgenic poplar. Up to date, examples of successful gene transformation of poplar are achieved with poplar organs using plant cell totipotency under aseptic condition. However, the difference among different varieties, different clones or individuals are relatively big, and the prior art methods often need a transgenic poplar variety and explants thereof. There is no plant regeneration technique. Exploration and research are needed on this field. This makes it unable to obtain transgenic plants of directional gene transformation for poplar needing an urgent transformation in a short term Therefore, one of the important tasks facing to the high and new technology field in today's rapid development in sciences, is how to build a simple, feasible, practicable and easy to operate transgenic regeneration technique for trees.
How to induce the production of non-embryonic tissue suitable for agrobacterium transformation and explore the suitable co-transformation conditions is one of the tasks of present agrobacterium mediated transformation of monocotyledon.